Richard K. Le Leu

Whey proteins as functional food ingredients

Putative anti-cancer activity of whey proteins has been investigated in an animal model to evaluate their potential role in disease prevention, and to contribute to a basis for their inclusion as ingredients in functional foods. Animal feeding trials have compared the efficacy of dietary whey proteins in retarding chemically induced colon cancer in a rat model of the disease. Dairy proteins, in particular whey protein, were found to be efficacious in retardation of colon cancer in young rats compared with other dietary proteins (meat, soy). The influence of dietary whey protein on development of colon cancer in mature rats has also been examined. Results similar to those with younger animals have been demonstrated, a finding that suggests age does not significantly alter the outcome. Efficacy of whey protein fractions has also been assessed. Preliminary results suggest that diets supplemented with lactoferrin or with β-lactoglobulin enhance protection against the development of putative tumor precursors (aberrant crypts) in the hind gut wall. The mechanism behind the apparent anti-cancer activity of dietary whey proteins in these studies may be related to their sulfur amino acid content, for which there is a high requirement in the rat, and hypothesised role in protecting DNA in methylated form. In a parallel study, a number of potential functional foods containing whey protein (flavored milk, pasta, ice cream, dessert pudding, muesli, and savory dip) have been developed in preparation for human clinical trials. The foods containing whey protein were generally highly acceptable in taste trials. These products are expected to be suitable as delivery vehicles for dietary whey protein in studies aimed at substantiating the human health benefits of this protein source.

A human, double-blind, placebo-controlled, crossover trial of prebiotic, probiotic, and synbiotic supplementation: effects on luminal, inflammatory, epigenetic, and epithelial biomarkers of colorectal cancer

BACKGROUND Diet is an important factor in colorectal carcinogenesis; thus, dietary supplements may have a role in colorectal cancer prevention. OBJECTIVE The objective was to establish the relative luminal, epithelial, and epigenetic consequences of prebiotic, probiotic, and synbiotic dietary supplementation in humans. DESIGN This was a randomized, double-blind, placebo-controlled, 4-wk crossover trial of resistant starch and Bifidobacterium lactis, either alone or as a combined synbiotic preparation, in 20 human volunteers. Rectal biopsy, feces, and serum samples were collected. The rectal mucosal endpoints were DNA methylation at 16 CpG island loci and LINE-1, epithelial proliferation (Ki67 immunohistochemistry), and crypt cellularity. The fecal endpoints were short-chain fatty acid concentrations, pH, ammonia, and microbiological profiles (by denaturing gradient gel electrophoresis and sequencing). Serum endpoints were a panel of cytokines and high-sensitivity C-reactive protein. RESULTS Seventeen subjects completed the entire study. The synbiotic intervention fostered a significantly different fecal stream bacterial community than did either the prebiotic (P = 0.032) or the probiotic (P = 0.001) intervention alone, in part because of a greater proportion of patients harboring fecal Lachnospiraceae spp. These changes developed in the absence of any significant differences in fecal chemistry. There were no differences in epithelial kinetics. CONCLUSIONS This synbiotic supplementation with B. lactis and resistant starch, in the doses used, induced unique changes in fecal microflora but did not significantly alter any other fecal, serum, or epithelial variables. This trial was registered in the Australian New Zealand Clinical Trials Registry at www.anzctr.org.au as ACTRN012606000115538.

Histone deacetylase inhibition in colorectal cancer cells reveals competing roles for members of the oncogenic miR-17-92 cluster.

Diet‐derived butyrate, a histone deacetylase inhibitor (HDI), decreases proliferation and increases apoptosis in colorectal cancer (CRC) cells via epigenetic changes in gene expression. Other HDIs such as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) have similar effects. This study examined the role of microRNAs (miRNAs) in mediating the chemo‐protective effects of HDIs, and explored functions of the oncogenic miR‐17‐92 cluster. The dysregulated miRNA expression observed in HT29 and HCT116 CRC cells could be epigenetically altered by butyrate, SAHA and TSA. These HDIs decreased expression of miR‐17‐92 cluster miRNAs (P < 0.05), with a corresponding increase in miR‐17‐92 target genes, including PTEN, BCL2L11, and CDKN1A (P < 0.05). The decrease in miR‐17‐92 expression may be partly responsible for the anti‐proliferative effects of HDIs, with introduction of miR‐17‐92 cluster miRNA mimics reversing this effect and decreasing levels of PTEN, BCL2L11, and CDKN1A (P < 0.05). The growth effects of HDIs may be mediated by changes in miRNA activity, with down‐regulation of the miR‐17‐92 cluster a plausible mechanism to explain some of the chemo‐protective effects of HDIs. Of the miR‐17‐92 cluster miRNAs, miR‐19a and miR‐19b were primarily responsible for promoting proliferation, while miR‐18a acted in opposition to other cluster members to decrease growth. NEDD9 and CDK19 were identified as novel miR‐18a targets and were shown to be pro‐proliferative genes, with RNA interference of their transcripts decreasing proliferation in CRC cells. This is the first study to identify competing roles for miR‐17‐92 cluster members, in the context of HDI‐induced changes in CRC cells.

Suppression of azoxymethane-induced colon cancer development in rats by dietary resistant starch

Resistant starch is a complex carbohydrate that reaches the colon where it can be fermented by the colonic microflora resulting in production of short chain fatty acids (SCFA), in particular butyrate. RS effects on colorectal tumourigenesis are contrasting and protection remains controversial. Butyrate has an important role as the preferred metabolic fuel and regulator of colonocyte proliferation, differentiation and apoptosis and may play a role in cancer prevention. Thus variation in butyrate production from different substrates might explain the variation in effect of RS. This study evaluated the hypothesis that feeding dietary resistant starch (as high amylose maize starch) would protect against azoxymethane (AOM)-colon carcinogenesis and favourably influence the colonic luminal environment. Male Sprague-Dawley rats (n = 90 were provided one of three diets: Control (without added dietary fibre or RS), 10% HAS (contained 100 g/kg raw high amylose maize starch) or 20% HAS (contained 200 g/kg high amylose maize starch). Rats were fed their experimental diets for 4 weeks after which they were injected with AOM (15 mg/kg) during the fifth and six week. Colons were resected (25 weeks post second injection) for evaluation of tumour formation, apoptosis, proliferating cell nuclear antigen (PCNA) labelling index and short chain fatty acid levels. Feeding resistant starch significantly reduced the incidence (P

Suppression of Colorectal Oncogenesis by Selenium-Enriched Milk Proteins: Apoptosis and K-ras Mutations

The chemical form and bioavailability of dietary selenium may influence its protectiveness against colorectal cancer. Selenium is readily incorporated into milk proteins by feeding cows with selenized-yeast. This study examined whether a dairy source of organic selenium (as milk proteins) is more effective than a yeast source at inhibiting oncogenesis in carcinogen-treated mice and whether it regulates the homeostatic response to carcinogen-induced DNA damage. Dietary interventions are as follows: selenium-enriched milk protein isolate (Tatura-Bio Se; 0.5 or 1 ppm selenium) or milk protein control and selenized-yeast (Sel-Plex; 1 or 4 ppm selenium) with casein or casein alone as control. After 4 weeks on diet, mice received a single azoxymethane (10 mg/kg) injection to induce mutations and were killed 6 hours later. Measures were as follows: plasma selenium, cell proliferation, and acute apoptotic response to azoxymethane (AARGC). Separate groups of mice on the same diets were given 4 weekly azoxymethane (15 mg/kg) injections to induce oncogenesis. Mice were killed 6 or 30 weeks after the last azoxymethane injection. Measures were as follows: aberrant crypt foci (ACF), cancers, and K-ras mutations. Dairy-selenium at 1 ppm significantly suppressed ACF and cancers, whereas yeast-selenium at an equivalent selenium intake had no effect. Dairy-selenium significantly increased plasma selenium levels and AARGC, and reduced cell proliferation and frequency of K-ras mutations in ACF relative to an equivalent dose of selenium from yeast. Selenium-enriched milk protein isolate is superior to selenized-yeast in terms of its bioavailability and capacity to suppress oncogenesis. Suppression may be a consequence of enhanced apoptotic deletion of azoxymethane-induced DNA lesions and the subsequent reduction in frequency of K-ras mutations.

The influence of dietary proteins on colon cancer risk

Experimental evidence is accumulating from animal models and in vitro data which shows that dietary proteins can influence cancer expression, some having a promotional influence, others a preventative effect relative to an arbitrarily established standard diet. This result will to a degree be determined by the nature of the cancer model under study. Dairy proteins have been shown to be relatively protective when compared with defatted soybean meal and cooked red meat in the rat dimethylhydrazine-induced (DMH) colon cancer model. Some epidemiological evidence supports these experimental observations. Both protein and fat appear to be influencing outcome, with potential for interactive effects. A number of possible mechanisms have been postulated as to how these proteins and closely associated factors could be influencing colon cancer risk, an area that deserves more investigation. Combinations of foods such as dairy foods with cereals and/or probiotic bacteria provide potentially interesting alliances in reducing colon cancer risk. The combining of relatively protective agents deserves more investigation as to its potential, in devising functional foods and diets worthy of further evaluation, in animal models of cancer, and human intervention studies using relevant endpoint markers.

A comparative study of the influence of differing barley brans on DMH-induced intestinal tumours in male Sprague-Dawley rats.

The influence of barley brans on the incidence and burden of intestinal tumours in rats induced by 1,2‐dimethylhydrazine (DMH) was studied in a 7 month feeding experiment. The basic diet was American Institute of Nutrition (AIN) 76 modified by adjustment to 20% fat and 40% starch; brans were added so as to supply 5% dietary fibre. The barley brans studied were commercial barley bran (BB1; 13.0% dietary fibre) from the aleurone/subaleurone layer, outerlayer barley bran (BB2) including the germ (25.5% dietary fibre) and spent barley grain bran (SBG; a by‐product of the brewery and including the hull; 47.7% dietary fibre). They were compared with wheat bran (WB; 44% dietary fibre) and cellulose (or control; 98% dietary fibre). Commercial barley bran and wheat bran were most effective in reducing tumour incidence and burden. The incidence of tumours fell significantly from 70% (BB2) and 50% (SBG) to 10% (BB1) and 20% (WB) and tumour burden and tumour mass index (TMI) were also reduced by similar orders of magnitude. There were significantly higher short chain fatty acid (SCFA) concentrations in WB‐ and BBl‐fed rat faecal pellets relative to cellulose‐ and BB2‐fed rat faeces; butyrate, in particular, was affected. Regression analysis of butyrate against tumour incidence showed a trend (r=‐0.898; P=0.055), but the concentration of butyrate alone could not account for the reduction in tumour incidence observed. In a second experiment, when two brans (BB1 and SBG) were introduced after DMH dosing, there were higher incidences and burdens of tumours, indicating that protection by such brans was not as effective under these circumstances. Commercially available barley bran and wheat bran appear to significantly reduce tumour incidence and burden in this model relative to other brans, influencing both the initiatory as well as promotional stages of chemically induced carcinogenesis.

Butyrylated starch intake can prevent red meat induced O6-methyl-2-deoxyguanosine adducts in human rectal tissue : a randomised clinical trial

Epidemiological studies have identified increased colorectal cancer (CRC) risk with high red meat (HRM) intakes, whereas dietary fibre intake appears to be protective. In the present study, we examined whether a HRM diet increased rectal O6-methyl-2-deoxyguanosine (O6MeG) adduct levels in healthy human subjects, and whether butyrylated high-amylose maize starch (HAMSB) was protective. A group of twenty-three individuals consumed 300 g/d of cooked red meat without (HRM diet) or with 40 g/d of HAMSB (HRM+HAMSB diet) over 4-week periods separated by a 4-week washout in a randomised cross-over design. Stool and rectal biopsy samples were collected for biochemical, microbial and immunohistochemical analyses at baseline and at the end of each 4-week intervention period. The HRM diet increased rectal O6MeG adducts relative to its baseline by 21 % (P< 0·01), whereas the addition of HAMSB to the HRM diet prevented this increase. Epithelial proliferation increased with both the HRM (P< 0·001) and HRM+HAMSB (P< 0·05) diets when compared with their respective baseline levels, but was lower following the HRM+HAMSB diet compared with the HRM diet (P< 0·05). Relative to its baseline, the HRM+HAMSB diet increased the excretion of SCFA by over 20 % (P< 0·05) and increased the absolute abundances of the Clostridium coccoides group (P< 0·05), the Clostridium leptum group (P< 0·05), Lactobacillus spp. (P< 0·01), Parabacteroides distasonis (P< 0·001) and Ruminococcus bromii (P< 0·05), but lowered Ruminococcus torques (P< 0·05) and the proportions of Ruminococcus gnavus, Ruminococcus torques and Escherichia coli (P< 0·01). HRM consumption could increase the risk of CRC through increased formation of colorectal epithelial O6MeG adducts. HAMSB consumption prevented red meat-induced adduct formation, which may be associated with increased stool SCFA levels and/or changes in the microbiota composition.

Inhibition by Resistant Starch of Red Meat-Induced Promutagenic Adducts in Mouse Colon

Population studies have shown that high red meat intake may increase colorectal cancer risk. Our aim was to examine the effect of different amounts and sources of dietary protein on induction of the promutagenic adduct O6-methyl-2-deoxyguanosine (O6MeG) in colonocytes, to relate these to markers of large bowel protein fermentation and ascertain whether increasing colonic carbohydrate fermentation modified these effects. Mice (n = 72) were fed 15% or 30% protein as casein or red meat or 30% protein with 10% high amylose maize starch as the source of resistant starch. Genetic damage in distal colonocytes was detected by immunohistochemical staining for O6MeG and apoptosis. Feces were collected for measurement of pH, ammonia, phenols, p-cresol, and short-chain fatty acids (SCFA). O6MeG and fecal p-cresol concentrations were significantly higher with red meat than with casein (P < 0.018), with adducts accumulating in cells at the crypt apex. DNA adducts (P < 0.01) and apoptosis (P < 0.001) were lower and protein fermentation products (fecal ammonia, P < 0.05; phenol, P < 0.0001) higher in mice fed resistant starch. Fecal SCFA levels were also higher in mice fed resistant starch (P < 0.0001). This is the first demonstration that high protein diets increase promutagenic adducts (O6MeG) in the colon and dietary protein type seems to be the critical factor. The delivery of fermentable carbohydrate to the colon (as resistant starch) seems to switch from fermentation of protein to that of carbohydrate and a reduction in adduct formation, supporting previous observations that dietary resistant starch opposes the mutagenic effects of dietary red meat. Cancer Prev Res; 4(11); 1920–8. ©2011 AACR.

Manipulation of the gut microbiota using resistant starch is associated with protection against colitis-associated colorectal cancer in rats

This study evaluated whether dietary resistant starch (RS) and green tea extract (GTE), which have anti-inflammatory and anticancer properties, protect against colitis-associated colorectal cancer (CAC) using a rat model, also investigated potential mechanisms of action of these agents including their effects on the gut microbiota. Rats were fed a control diet or diets containing 10% RS, 0.5% GTE or a combination of the two (RS + GTE). CAC was initiated with 2 weekly azoxymethane (AOM) injections (10mg/kg) followed by 2% dextran sodium sulphate in drinking water for 7 days after 2 weeks on diets. Rats were killed 20 weeks after the first AOM. Colon tissues and tumours were examined for histopathology by H&E, gene/protein expression by PCR and immunohistochemistry and digesta for analyses of fermentation products and microbiota populations. RS and RS + GTE (but not GTE) diets significantly (P< 0.05) decreased tumour multiplicity and adenocarcinoma formation, relative to the control diet. Effects of RS + GTE were not different from RS alone. RS diet caused significant shifts in microbial composition/diversity, with increases in Parabacteroides, Barnesiella, Ruminococcus, Marvinbryantia and Bifidobacterium as primary contributors to the shift. RS-containing diets increased short chain fatty acids (SCFA) and expression of the SCFA receptor GPR43 mRNA, and reduced inflammation (COX-2, NF-kB, TNF-α and IL-1β mRNA) and cell proliferation P< 0.05. GTE had no effect. This is the first study that demonstrates chemopreventive effects of RS (but not GTE) in a rodent CAC model, suggesting RS might have benefit to patients with ulcerative colitis who are at an increased risk of developing CRC.